EP1405919B1 - Verfahren zur elektronischen Überwachung und Aufzeichnung von Zellkulturen - Google Patents

Verfahren zur elektronischen Überwachung und Aufzeichnung von Zellkulturen Download PDF

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Publication number
EP1405919B1
EP1405919B1 EP03025616A EP03025616A EP1405919B1 EP 1405919 B1 EP1405919 B1 EP 1405919B1 EP 03025616 A EP03025616 A EP 03025616A EP 03025616 A EP03025616 A EP 03025616A EP 1405919 B1 EP1405919 B1 EP 1405919B1
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EP
European Patent Office
Prior art keywords
electrically conductive
pair
alternating current
current voltage
elongated pins
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EP03025616A
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English (en)
French (fr)
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EP1405919A1 (de
Inventor
Patricia J. Malin
Kenneth Richard Wada
Oskar Werner Huber
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CellStat Technologies Inc
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CellStat Technologies Inc
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Priority to DE1992633731 priority Critical patent/DE69233731T2/de
Priority claimed from EP92916640A external-priority patent/EP0655086B1/de
Publication of EP1405919A1 publication Critical patent/EP1405919A1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48735—Investigating suspensions of cells, e.g. measuring microbe concentration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Definitions

  • the present invention relates generally to methods for monitoring the growth of cells while being cultured and, more particularly, to such methods that employ an electrical current for monitoring the culture.
  • PCT Patent Cooperation Treaty
  • PCT/US91/02320 filed 3 April 1991 , published 17/October 1991, and entitled "An Electronic Technique of Identifying an Effective Drug for Treating a Cancer Patient” discloses a system for monitoring and recording cell cultures.
  • This PCT patent application claims priority from a similarly titled United States patent application no. 07/503,791 filed 3 April 1990 , that discloses a similar cell culture monitoring and recording system.
  • the system disclosed in these patent applications monitors and records cells being cultured in a conventional laboratory tray.
  • the tray has a plurality of separate wells with each well being open at one end.
  • This system includes a lid that is disposed on top of the tray to cover the open ends of the tray's wells.
  • a printed circuit board Secured to the top surface of the lid is a printed circuit board that has a plurality of individual electrically conductive traces formed on one surface. One end of each trace is adapted to mate with and engage a printed circuit board connector.
  • a non-reactive, stainless steel pin that penetrates both the printed circuit board and the lid. The placement of the traces on the printed circuit board with respect to the lid arranges the pins in pairs so that when the lid is placed on the tray each pair of pins is inserted through the open end of and extends into a different well in the tray.
  • This system also includes a printed circuit board connector that mates with the traces of the printed circuit board and through which one pin of every pair is electrically connected to a common potential.
  • An electronic data acquisition board is electrically coupled to the printed circuit board connector.
  • the electronic data acquisition board includes a voltage source for applying an electrical voltage across a pair of the pins.
  • a plurality of well switches also included in the electronic data acquisition board, selects one particular pair of the pins to receive the voltage supplied by the voltage source.
  • the electronic data acquisition board preferably applies an alternating current voltage of approximately 10 millivolts peak-to-peak across the pair of pins. This alternating current voltage preferably has a frequency of approximately 400 Hz that is not harmonically related to 60 Hz.
  • An amplifier also included in the electronic data acquisition board, has an input that receives the voltage applied across the pair of pins selected by the well switches. From its output, the amplifier transmits a signal that responds to the voltage present at the amplifier's input to a sample-and-hold circuit.
  • the sample-and-hold circuit receives the output signal from the amplifier and, in turn, transmits a signal responsive to that signal.
  • This cell culture monitoring and recording system also includes a computer system that itself includes a measurement input/output circuit.
  • the measurement input/output circuit includes means for supplying signals to the electronic data acquisition board for specifying a particular pair of pins to be selected by the switches.
  • the measurement input/output circuit also includes an analog-to-digital converter that receives and digitizes the signal from the sample-and-hold amplifier to produce a digital number.
  • the computer system includes a means for storing them as raw data, and means for analyzing the raw data and graphically displaying the results of such analysis.
  • An object of the present invention is to provide an improved method for monitoring and recording cell cultures.
  • Another object of the present invention is to provide a cell culture monitoring and recording method that achieves higher stability.
  • Another object of the present invention is to provide a cell culture monitoring and recording method that more precisely measures electrical conductivity of cells being cultured.
  • Another object of the present invention is to provide a cell culture monitoring and recording method that does not harm cells during culture.
  • Another object of the present invention is to provide a cell culture monitoring and recording method that more effectively presents test results.
  • This cell culture monitoring and recording method allows the use of a lid having more reliable electrical connections between non-reactive pins thereof and electrically conductive traces on a printed circuit board included therein.
  • the present invention allows the use of an improved lid having a plurality of individual pin sockets that are interposed between traces on a printed circuit board and electrically conductive pins that, in use, extend into cell growth media held in wells of a standard laboratory tray.
  • Using such pin sockets provides more reliable electrical connection between the non-reactive, electrically conductive pins and the traces on the printed circuit board.
  • the improved method also determines a delay period used in measuring electrical conductivity between a pair of pins while applying an alternating current electrical potential across the pair of pins to monitor cell growth.
  • This delay period in measuring electrical conductivity increases measurement stability.
  • a low, safe voltage is initially applied across a pair of pins prior to monitoring cell growth, and then the voltage is successively increased until reaching a pre-established voltage.
  • This method for applying the pre-established voltage avoids the possibility of exposing the cells to an excessively large electrical potential.
  • an electrical conductivity measured for a reference well that holds only cell growth media without cells is substracted from that measured for wells that hold both cell growth media and cells.
  • FIGs. 1 and 2 illustrate alternative views of a lid 10 for use in the present invention.
  • the lid 10 includes a cover 12 adapted to be positioned over a laboratory tray 14 in the manner illustrated in FIG. 3 .
  • the laboratory tray 14 includes a plurality of wells 16, each well 16 having an open end 18.
  • Each of the wells 16 is adapted for holding a quantity of cell growth media in which cells may be cultured.
  • the cover 12 of the lid 10 is positioned over the laboratory tray 14, the cover 12 blocks the open ends 18 of the wells 16.
  • a printed circuit board 22 that is secured to the cover 14.
  • Various alternative techniques may be employed for securing the printed circuit board 22 to the cover 14. For example, machine screws (not illustrated in any of the FIGs.) may extend through matching apertures formed through the printed circuit board 22 and cover 14, the machine screws being secured there by mating nuts threaded thereon (not illustrated in any of the FIGs.).
  • the printed circuit board 22 may be secured to the cover 14 by an adhesive material disposed between their juxtaposed surfaces.
  • the printed circuit board 22 includes a plurality of individual electrically conductive traces 24 formed on its surface as illustrated in FIGs. 1-4 . As illustrated in FIG. 1-3 , one end of each trace 24 is adapted to mate with and engage a printed circuit board connector (not illustrated in any of the FIGs.). An aperture 26 is formed through the printed circuit board 22 near the other end of each trace 24 to receive a first end 27 of a pin socket 28.
  • the pin sockets 28, preferably model SC 5P1 ⁇ GG manufactured by Samtec, Inc. of New Albany, Indiana, are respectively electrically coupled and secured to the traces 24 by solder 32.
  • a circular aperture 34 formed through the cover 12 concentric with the pin socket 28, receives a second end 36 of the pin socket 28.
  • each pin socket 28 receives and retains an elongated pin 38 preferably formed from a non-reactive, electrically conductive material such as gold or stainless steel wire 0.508 mm (0.020 inches) in diameter.
  • Each of the pin sockets 28 included in the lid 10 preferably forms a gas-tight seal with the pin 38 inserted thereinto.
  • the placement of the traces 24 on the printed circuit board 22 arranges the pin sockets 28 and pins 38 into pairs.
  • the combined length of each pin socket 28 and the pin 38 received thereinto penetrates both the printed circuit board 22 and the cover 12 with the ends of each pair of pins 38 furthest from the pin socket 28 being insertable through the open end 18 of a well 16 in the laboratory tray 14 when the lid 10 is positioned thereover as illustrated in FIG. 4 .
  • So positioning the lid 10 on the laboratory tray 14 disposes each pair of pins 38 so they may extend into cell growth media held in the well 16.
  • the end of the pins 38 furthest from the pin sockets 28 must not contact the end of the well 16 furthest from its open end 18, and the quantity of cell growth media held in the well 16 must be sufficiently small that the media does not contact the pin sockets 28.
  • the lid 10 Before the system may be used to monitor and record cultured cells, the lid 10 must be sterilized so it will not contaminate the cell culture. However, sterilizing the lid 10 must be performed properly to avoid interrupting electrical conductivity between the pins 38 and the traces 24, particularly at the gas-tight engagement between the pin socket 28 and the pin 38.
  • One procedure that has been successfully applied for sterilizing the lids 10 is to immerse the lid 10 in 70% isopropyl alcohol or ethanol, and then to dry the lid 10 in a laminar flow hood while exposing it to ultraviolet light.
  • FIG. 5 is a block diagram in accordance with the preferred embodiment of the present invention depicting a portion of the electronic circuit for applying the potential across a pair of pins 38 and for monitoring cultured cells.
  • the computer program executed by the computer system (omitted from FIG.
  • the measurement input/output board 46 of the present embodiment is preferably a MetraByte DAS-8 Data Acquisition and Control Board marketed by Keithley Metrabyte Corporation of Taunton, Massachusetts.
  • the data acquisition board 44 includes a programmable voltage source 48.
  • the programmable voltage source 48 includes an alternating current generator 52 that produces a 370 Hz ⁇ 20%, 10 volt peak-to-peak sine wave signal.
  • the output signal produced by the alternating current generator 52 is transmitted to a programmable attenuator 54 also included in the programmable voltage source 48.
  • Digital excitation level control- signals supplied from the computer system to the programmable attenuator 54 via excitation level control signal lines 56 permit adjustment of the peak-to-peak voltage of the alternating current supplied to a first terminal 57 of a 20. 04 K ohm resistor 58.
  • a second terminal 59 of the resistor 58 connects to a bank of switches 62.
  • One of the switches 62 is selected by the computer system, as disclosed in the patent applications identified above, for applying the alternating current voltage supplied by the programmable voltage source 48 to a pair of pins 38 that extend into the well 16 being monitored.
  • the alternating current voltage applied across a pair of pins 38 is also supplied to the input of the programmable gain amplifier 42.
  • the gain of the programmable gain amplifier 42 may be adjusted by control signals supplied from the computer system via gain control signal lines 64.
  • the output signals from the programmable attenuator 54, and from the programmable gain amplifier 42 are both supplied to a multiplexer 66.
  • Control signals supplied from the computer system to the multiplexer 66 via multiplexer control signal lines 68 select one of these three signals for application to an input of a sample-and-hold amplifier 70 included in the measurement input/output board 46.
  • the output signal from the sample-and-hold amplifier 70 is supplied to the input of an analog-to-digital converter 72 also included in the measurement input/output board 46.
  • the 10 volt peak-to-peak output signal from the alternating current generator 52 is also supplied to the input of a comparator 74.
  • the output signal from the comparator 74 changes state each time the alternating current voltage produced by the alternating current generator 52 passes through zero volts.
  • the output signal from the comparator 74 is in one state, and while that voltage has a potential less than zero volts, the output signal from the comparator 74- is in its other state.
  • the output signal from the comparator 74 is supplied to a programmable timer 76 included in the measurement input/output board 46.
  • the frequency of the alternating current voltage may change over time, it has proven beneficial to the accurate and repeatable operation of the cell culture monitoring and recording system to adjust the instant at which the sample-and-hold amplifier 70 samples the output signal from the programmable gain amplifier 42 to match the frequency of the alternating current voltage produced by the programmable voltage source 48.
  • FIG. 6 depicts a sinusoidal alternating current waveform 82 for the voltage present at the output of the alternating current generator 52 together with the a digital waveform 84 of the output signal produced by the comparator 74.
  • the computer program executed by the computer system executes a procedure for establishing a delay period ("D"), illustrated in FIG. 6 , beginning at the instant at which the sine waveform 82 in changing from a positive potential to a negative potential has a potential of zero volts, and the instant at which the sine waveform 82 has its immediately subsequent maximum positive value.
  • D delay period
  • the computer program uses the output signal from the comparator 74 in the data acquisition board 44 together with the programmable timer 76 included in the measurement input/output board 46 to determine the duration of one period of the sine waveform 82.
  • the computer program then establishes the delay period D at three-fourths of one period of the sine waveform 82. Having determined a proper delay period D, the computer program then loads that delay period into the programmable timer 76 so that all subsequent measurements of the electrical potential across a pair of pins 38 will occur precisely at the instant that the voltage supplied to the first terminal 57 of the resistor 58 reaches its maximum value.
  • the programmable timer 76 begins measuring each delay period at the instant at which the sine waveform 82 in changing from a positive potential to a negative potential has a potential of zero volts, i.e. immediately after the digital value of the output signal from the comparator 74 supplied to the programmable timer 76 changes from 0 to 1.
  • the programmable timer 76 immediately causes the sample-and-hold amplifier 70 to sample and hold the voltage of the signal supplied from the output of the programmable gain amplifier 42 via the multiplexer 66 as illustrated in the waveform 88 depicted in FIG. 6 .
  • the programmable timer 76 also causes the analog-to-digital converter 72 to convert the voltage of the analog signal received from the sample-and-hold amplifier 70 into a digital number. Subsequently, as described in the patent applications identified above, this digital number is transferred from the measurement input/output board 46 to the computer system for storage as raw data suitable for subsequent analysis and graphic display.
  • the program executed by the computer system determines the present duration to be used for the delay period D when monitoring cell cultures, it then prepares the alternating current voltage to be applied from the second terminal 59 of the resistor 58 across a pair of pins 38 during such monitoring.
  • the computer program first adjusts the programmable attenuator 54 so a potential of approximately 10 millivolts is present at its output and at the first terminal 57 of the resistor 58.
  • the resistor 58 separates its first terminal 57 from its second terminal 59, and because any cell growth media held in the well 16 provides some electrical conductivity between the pair of pins 38 inserted therein, initially the voltage at the second terminal 59 and across a pair of pin 38 must be less than the value of 10 millivolts intended to be used in measuring the conductivity between a pair of pins 38.
  • the computer program then causes the multiplexer 66 to select the output signal from the programmable gain amplifier 42 for application to the input of the sample-and-hold amplifier 70, sets the gain of the programmable gain amplifier 42 so a peak voltage of 10 millivolts at the second terminal 59 of the resistor 58 will result in the analog-to-digital converter 72 producing a digital number that is approximately 83.3% of the full range of the analog-to-digital converter 72, and causes the bank of switches 62 to select a pair of pins 38 for application of the alternating current voltage.
  • the cell culture monitoring and recording system uses the previously determined delay period D and the measurement input/output board 46 to measure the peak alternating current voltage present at the second terminal 59 of the resistor 58 that is applied across the pair of pins 38. If the voltage at the second terminal 59 and across the pair of pins 38 is less than 5 millivolts, the computer program doubles the alternating current voltage produced by the programmable attenuator 54 repeatedly until the voltage measured at the second terminal 59 exceeds 5 millivolts.
  • the computer program Having thus applied and measured an alternating current voltage across the pair of pins 38 that exceeds 5 millivolts and knowing the setting for the programmable attenuator 54 which produces that voltage, the computer program then computes a new setting for the programmable attenuator 54 that will apply approximately a 10 millivolt alternating current voltage to the second terminal 59 and across the pair of pins 38, and then transmits control signals setting the attenuator 54 to the computed value.
  • the system is now prepared to monitor and record the electrical conductivity of the well 16.
  • the computer program first repetitively measures the voltage applied across the pair of pins 38 and at the second terminal 59 of the resistor 58. After collecting 16 successive values for this voltage, the computer program then computes an average of the 16 values using a box-car filter to obtain a single, average value for the voltage across the pair of pins 38.
  • the computer program uses the single value of the voltage across the pair of pins 38, the value of the voltage supplied by the programmable attenuator 54 to the first terminal 57 of the resistor 58, and the resistance of the resistor 58; the computer program then computes the conductivity of the cell growth media and cells, if any, between the pair of pins 38.
  • the computer program first stores the conductivity value for subsequent analysis and then proceeds to measure the conductivity between another pair of pins 38 extending into another well 16 in the laboratory tray 14.
  • the cell culture monitoring and recording system uses the procedures set forth above of first adjusting the alternating current voltage applied across the pair of pins 38, and then measuring and averaging the voltage applied across the pair of pins 38 extending into the well 16. This adjusting of the applied voltage and determining of cell conductivity is repeated over and over until a conductivity has been determined and stored for all wells 16 in the laboratory tray 14.
  • At least one of the wells 16 in the laboratory tray 14 must be a reference well 16 that holds only cell culture media without cells. Furthermore, this reference well 16 must be specifically so identified to the analysis computer program because that program uses the conductivity value for the reference well 16 in analyzing the conductivity for all the other wells 16.
  • analysis of the conductivity of a well 16 that held both cell growth media and cells included dividing the conductivity measured for the reference well 16 by the conductivity measured for the well 16 that held both the cell growth media and cells.
  • the electrical conductivity of the reference well 16 as the numerator of a fraction in analyzing the conductivity of a well that holds both cell growth media and cells
  • a triangular waveform alternating current voltage may be easily generated using a digital logic circuit, in a system employing such a waveform it would be unnecessary to directly measure, as described above, the delay period D. Rather the digital circuits used in generating the triangular waveform alternating current voltage could themselves directly produce signals for controlling the operation of the sample-and-hold amplifier 70 and the analog-to-digital converter 72. However, such a system for monitoring and recording cell cultures would merely employ a different, well known technique for determining the delay period for its alternating current voltage that is equal to an interval of time between the alternating current voltage having an instantaneous potential of zero volts and having an instantaneous potential equal to the maximum voltage of the alternating current voltage.
  • the method for monitoring and recording cell cultures according to the present invention may be applied for nondestructively measuring the kinetics of cell growth. It may also be applied to measure the response kinetics of target cell populations to hormones, growth factors, growth inhibitors, cytotoxins, and drugs that perturb cellular metabolism.
  • This method for monitoring and recording cell cultures is particularly useful in areas such as anticancer drug screening.
  • Conventional screening methods measure culture response following a fixed incubation time. Chemotherapeutic drugs which exhibit delayed action are often falsely identified as being inactive, while drugs which act very quickly may be indistinguishable from more slowly acting compounds.
  • the disclosed method for monitoring and recording cell cultures allows longer assay durations to identify drugs which exhibit a delayed effect, while also identifying drugs that act more rapidly.

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Claims (17)

  1. Verfahren zum elektronischen Überwachen und Aufzeichnen von in einer Laborschale mit mehreren getrennten Schächten kultivierten Zellen, wobei jeder der mehreren getrennten Schächten dazu geeignet ist, eine Menge eines Zellwachstumsmediums aufzunehmen, in dem Zellen kultiviert werden können, wobei das Verfahren die Schritte aufweist:
    a. Bereitstellen einer Laborschale mit mehreren getrennten Schächten, wobei jeder Schacht dazu geeignet ist, eine Menge eines Zellwachstumsmediums aufzunehmen, in dem Zellen kultiviert werden können;
    b. Anordnen jeweils eines Paars elektrisch leitfähiger länglicher Stifte durch ein offenes Ende jedes der Schächte in der Laborschale derart, dass sie sich in das darin aufgenommene Zellwachstumsmedium erstrecken;
    c. Zuführen einer Wechselspannung, die zwischen einem Paar der elektrisch leitfähigen länglichen Stifte selektiv angelegt werden soll, zum elektronischen Überwachen und Aufzeichnen der in der Laborschale kultivierten Zellen;
    d. Bestimmen einer Verzögerungszeit für die Wechselspannung vor der Auswahl eines Paars der elektrisch leitfähigen länglichen Stifte, zwischen denen die Wechselspannung angelegt werden soll, wobei die Verzögerungszeit einem Zeitintervall gleicht, das zu dem Zeitpunkt beginnt, zu dem die Wechselspannung, die über ein Stiftpaar angelegt wird, während einer Änderung von einem positiven zu einem negativen Potenzial einen Momentanwert von null Volt aufweist, und zu einem Zeitpunkt endet, zu dem die Wechselspannung einen unmittelbar folgenden Maximalwert erreicht; und
    e. Verwenden der vorangehend bestimmten Verzögerungszeit, nachdem ein Paar elektrisch leitfähiger länglicher Stifte ausgewählt wurde, an die die Wechselspannung angelegt werden soll, zum Messen der elektrischen Leitfähigkeit zwischen dem Paar elektrisch leitfähiger länglicher Stifte exakt zu dem Zeitpunkt, zu dem die Spannung ihren Maximalwert erreicht.
  2. Verfahren nach Anspruch 1, wobei die Wechselspannung eine symmetrische Wellenform hat und die Verzögerungszeit 3/4 einer Periode der Wechselspannung gleicht.
  3. Verfahren nach Anspruch 2, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung eine Frequenz von weniger als 1000 Hz hat.
  4. Verfahren nach Anspruch 2, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung eine Frequenz hat, die mit 60 Hz nicht harmonisch in Beziehung steht.
  5. Verfahren nach Anspruch 2, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung eine Frequenz zwischen 300 und 400 Hz hat.
  6. Verfahren nach Anspruch 5, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung eine Frequenz hat, die mit 60 Hz nicht harmonisch in Beziehung steht.
  7. Verfahren nach Anspruch 1, ferner mit dem Schritt zum Bereitstellen eines Deckels mit: einer Abdeckung, die dazu geeignet ist, auf der Laborschale angeordnet zu werden, um die offenen Enden der mehreren getrennten Schächte in der Laborschale zu verschließen; einer an einer Oberseite der Abdeckung befestigten Leiterplatte mit mehreren auf einer ihrer Oberflächen ausgebildeten elektrischen Leiterbahnen; und mehreren Stiftsockeln, deren Anzahl der Anzahl der auf der Leiterplatte ausgebildeten elektrischen Leiterbahnen gleicht, wobei die Stiftsockel jeweils mit einer auf der Leiterplatte ausgebildeten elektrischen Leiterbahn elektrisch verbunden und daran befestigt sind, und wobei jeder der Stiftsockel einen der elektrisch leitfähigen länglichen Stifte aufnimmt.
  8. Verfahren nach Anspruch 7, wobei jeder der Stiftsockel einen gasdichten Eingriff mit dem darin aufgenommenen elektrisch leitfähigen länglichen Stift bildet.
  9. Verfahren nach Anspruch 7, wobei die im Deckel angeordneten Stiftsockel sich nicht in das in den mehreren getrennten Schächten aufgenommene Zellwachstumsmedium erstrecken.
  10. Verfahren nach Anspruch 7, wobei die mehreren elektrisch leitfähigen länglichen Stifte aus einem nicht reaktionsfähigen elektrisch leitfähigen Material hergestellt sind.
  11. Verfahren nach Anspruch 10, wobei die mehreren elektrisch leitfähigen länglichen Stifte aus rostfreiem Stahl hergestellt sind.
  12. Verfahren nach Anspruch 7, wobei ein elektrisch leitfähiger länglicher Stift jedes Paars mit einem gemeinsamen Potenzial elektrisch verbunden ist.
  13. Verfahren nach Anspruch 1, ferner mit den Schritten:
    e. Auswählen eines ersten Paars elektrisch leitfähiger länglicher Stifte, die in einen Referenzschacht eingeführt sind, der nur ein Zellwachstumsmedium, aber keine Zellen enthält, zum Anlegen der Wechselspannung;
    f. Messen der elektrischen Leitfähigkeit zwischen dem in den Referenzschacht eingeführten ersten Paar elektrisch leitfähiger länglicher Stifte;
    g. Auswählen eines zweiten Paars elektrisch leitfähiger länglicher Stifte, die in einen Schacht eingeführt sind, der ein Zellwachstumsmedium mit Zellen enthält, zum Anlegen der Wechselspannung;
    h. Messen der elektrischen Leitfähigkeit zwischen dem Paar elektrisch leitfähiger länglicher Stifte, die in den Schacht eingeführt sind, der das Zellwachstumsmedium mit Zellen enthält;
    i. Subtrahieren der für den Referenzschacht gemessenen elektrischen Leitfähigkeit von der für den Schacht, der das Zellwachstumsmedium mit Zellen enthält, gemessenen elektrischen Leitfähigkeit.
  14. Verfahren nach Anspruch 1, wobei die zugeführte Wechselspannung anfangs kleiner ist als ein vorgegebener Sollwert, der für eine Messung der elektrischen Leitfähigkeit zwischen dem Paar elektrisch leitfähiger länglicher Stifte vorgesehen ist, wobei das Verfahren ferner die Schritte aufweist:
    e. Messen der zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegten Wechselspannung;
    f. Erhöhen der Größe der zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegten Wechselspannung, bis sie dem vorgegebenen Sollwert im Wesentlichen gleicht; und
    g. Messen der elektrischen Leitfähigkeit zwischen dem Paar elektrisch leitfähiger länglicher Stifte.
  15. Verfahren nach Anspruch 14, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung kleiner ist als 90 Millivolt.
  16. Verfahren nach Anspruch 15, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung kleiner ist als 50 Millivolt.
  17. Verfahren nach Anspruch 16, wobei die zwischen dem Paar elektrisch leitfähiger länglicher Stifte angelegte Wechselspannung kleiner ist als 20 Millivolt.
EP03025616A 1992-07-29 1992-07-29 Verfahren zur elektronischen Überwachung und Aufzeichnung von Zellkulturen Expired - Lifetime EP1405919B1 (de)

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US3743581A (en) * 1970-10-21 1973-07-03 Bactomatic Inc Microbiological detection apparatus
US4156180A (en) * 1977-06-24 1979-05-22 Bactomatic, Inc. Apparatus and method for detecting metabolic activity
US4230983A (en) * 1978-11-24 1980-10-28 Agro Sciences, Inc. Seed viability analyzer
GB2186366B (en) * 1986-02-06 1989-11-22 Metal Box Plc Improvements in or relating to apparatus for detecting micro-organisms
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